Delany Rodriguez

Migration of germline progenitor cells is directed by sphingosine-1-phosphate signalling in a basal chordate

The colonial ascidian Botryllus schlosseri continuously regenerates entire bodies in an asexual budding process. The germ line of the newly developing bodies is derived from migrating germ cell precursors, but the signals governing this homing process are unknown. Here we show that germ cell precursors can be prospectively isolated based on expression of aldehyde dehydrogenase and integrin alpha-6, and that these cells express germ cell markers such as vasa, pumilio and piwi, as well as sphingosine-1-phosphate receptor. In vitro, sphingosine-1-phosphate (S1P) stimulates migration of germ cells, which depends on integrin alpha-6 activity. In vivo, S1P signalling is essential for homing of germ cells to newly developing bodies. S1P is generated by sphingosine kinase in the developing germ cell niche and degraded by lipid phosphate phosphatase in somatic tissues. These results demonstrate a previously unknown role of the S1P signalling pathway in germ cell migration in the ascidian Botryllus schlosseri.

Analysis of the basal chordate Botryllus schlosseri reveals a set of genes associated with fertility

BackgroundGonad differentiation is an essential function for all sexually reproducing species, and many aspects of these developmental processes are highly conserved among the metazoa. The colonial ascidian, Botryllus schlosseri is a chordate model organism which offers two unique traits that can be utilized to characterize the genes underlying germline development: a colonial life history and variable fertility. These properties allow individual genotypes to be isolated at different stages of fertility and gene expression can be characterized comprehensively.ResultsHere we characterized the transcriptome of both fertile and infertile colonies throughout blastogenesis (asexual development) using differential expression analysis. We identified genes (as few as 7 and as many as 647) regulating fertility in Botryllus at each stage of blastogenesis. Several of these genes appear to drive gonad maturation, as they are expressed by follicle cells surrounding both testis and oocyte precursors. Spatial and temporal expression of differentially expressed genes was analyzed by in situ hybridization, confirming expression in developing gonads.ConclusionWe have identified several genes expressed in developing and mature gonads in B. schlosseri. Analysis of genes upregulated in fertile animals suggests a high level of conservation of the mechanisms regulating fertility between basal chordates and vertebrates.

Whole-mount fluorescent in situ hybridization staining of the colonial tunicate Botryllus schlosseri

Botryllus schlosseri is a colonial ascidian with characteristics that make it an attractive model for studying immunology, stem cell biology, evolutionary biology, and regeneration. Transcriptome sequencing and the recent completion of a draft genome sequence for B. schlosseri have revealed a large number of genes, both with and without vertebrate homologs, but analyzing the spatial and temporal expression of these genes in situ has remained a challenge. Here, we report a robust protocol for in situ hybridization that enables the simultaneous detection of multiple transcripts in whole adult B. schlosseri using Tyramide Signal Amplification in conjunction with digoxigenin‐ and dinitrophenol‐labeled RNA probes. Using this protocol, we have identified a number of genes that can serve as markers for developing and mature structures in B. schlosseri, permitting analysis of phenotypes induced in loss‐of‐function experiments. genesis 53:194–201, 2015.

Aging in the colonial chordate, Botryllus schlosseri

What mechanisms underlie aging? One theory, the wear-and-tear model, attributes aging to progressive deterioration in the molecular and cellular machinery which eventually lead to death through the disruption of physiological homeostasis. The second suggests that life span is genetically programmed, and aging may be derived from intrinsic processes which enforce a non-random, terminal time interval for the survivability of the organism. We are studying an organism that demonstrates both properties: the colonial ascidian, Botryllus schlosseri. Botryllus is a member of the Tunicata, the sister group to the vertebrates, and has a number of life history traits which make it an excellent model for studies on aging. First, Botryllus has a colonial life history, and grows by a process of asexual reproduction during which entire bodies, including all somatic and germline lineages, regenerate every week, resulting in a colony of genetically identical individuals. Second, previous studies of lifespan in genetically distinct Botryllus lineages suggest that a direct, heritable basis underlying mortality exists that is unlinked to reproductive effort and other life history traits. Here we will review recent efforts to take advantage of the unique life history traits of B. schlosseri and develop it into a robust model for aging research.

Gonad development and hermaphroditism in the ascidian Botryllus schlosseri

The colonial ascidian Botryllus schlosseri is an ideal model organism for studying gonad development and hermaphroditism. B. schlosseri has been reared in laboratories for over half a century, and its unique biology allows investigators to probe the processes of germ cell migration and gonad formation, resorption, and regeneration. Following metamorphosis, colonies of B. schlosseri show a synchronized and sequential fertility program that, under standard laboratory conditions, begins with a juvenile stage with no visible gonads and subsequently develops testes at 9 weeks followed later by the production of oocytes—thus resulting in hermaphroditic individuals. The timing of oocyte production varies according to the season, and adult B. schlosseri colonies can cycle among infertile and both male and hermaphrodite fertile states in response to changing environmental conditions. Thus, these acidians are amenable to studying the molecular mechanisms controlling fertility, and recent genomic and transcriptomic databases are providing insight to the key genes involved. Here, we review the techniques and approaches developed to study germ cell migration and gonad formation in B. schlosseri, and include novel videos showing processes related to oocyte ovulation and sperm discharge. In the future, this valuable invertebrate model system may help understand the mechanisms of gonad development and regeneration in a chordate. Mol. Reprod. Dev. 84: 158–170, 2017.

Colonial ascidians as model organisms for the study of germ cells, fertility, whole body regeneration, vascular biology and aging

Colonial ascidians are marine invertebrate chordates that are the closest invertebrate relative to the vertebrates. Colonies of Botryllus schlosseri undergo a continuous asexual reproduction process that involves the regeneration of entire new bodies, which include all somatic and germline tissues. This adult regenerative process depends on signaling pathways known to regulate the formation of tissues and organs during embryonic development. The formation of gonads within regenerated bodies depends on migration and homing of germ cell precursors to niches within the developing bodies, and Botryllus colonies can cycle between fertile and infertile states. The vasculature that connects all individuals within the colony is highly regenerative, and is a valuable tool for the study of angiogenesis in adult blood vessels. The tremendous regenerative capacity of the vasculature even results in regeneration of entire new bodies solely from fragments of blood vessels upon surgical removal of all bodies. The mechanism underlying this regeneration of whole bodies is not well understood, but appears to depend on proliferation of circulating, blood borne cells. Because of all of these features, colonial ascidians are ideal model organisms for the study of germ cell migration, fertility, vascular biology and regeneration.

Molecular evolution and in vitro characterization of Botryllus histocompatibility factor

Botryllus schlosseri is a colonial ascidian with a natural ability to anastomose with another colony to form a vascular and hematopoietic chimera. In order to fuse, two individuals must share at least one allele at the highly polymorphic fuhc locus. Otherwise, a blood-based inflammatory response will occur resulting in a melanin scar at the sites of interaction. The single-locus genetic control of allorecognition makes B. schlosseri an attractive model to study the underlying molecular mechanisms. Over the past decade, several candidate genes involved in allorecognition have been identified, but how they ultimately contribute to allorecognition outcome remains poorly understood. Here, we report our initial molecular characterization of a recently identified candidate allodeterminant called Botryllus histocompatibility factor (bhf). bhf, both on a DNA and protein level, is the least polymorphic protein in the fuhc locus studied so far and, unlike other known allorecognition determinants, does not appear to be under any form of balancing or directional selection. Additionally, we identified a second isoform through mRNA-Seq and an EST assembly library which is missing exon 3, resulting in a C-terminally truncated form. We report via whole-mount fluorescent in situ hybridization that a subset of cells co-express bhf and cfuhcsec. Finally, we observed BHF’s localization in HEK293T at the cytoplasmic side of the plasma membrane in addition to the nucleus via a nuclear localization signal. Given the localization data thus far, we hypothesize that BHF may function as a scaffolding protein in a complex with other Botryllus proteins, rather than functioning as an allorecognition determinant.

In vivo manipulation of the extracellular matrix induces vascular regression in a basal chordate

Remodeling of the extracellular matrix plays an important role in vascular homeostasis in the basal chordate Botryllus schlosseri, whose large, transparent, extracorporeal vascular network makes it an ideal system for studies of vascular mechanotransduction.

The Biology of the Extracorporeal Vasculature of Botryllus schlosseri

The extracorporeal vasculature of the colonial ascidian Botryllus schlosseri plays a key role in several biological processes: transporting blood, angiogenesis, regeneration, self-nonself recognition, and parabiosis. The vasculature also interconnects all individuals in a colony and is composed of a single layer of ectodermally-derived cells. These cells form a tube with the basal lamina facing the lumen, and the apical side facing an extracellular matrix that consists of cellulose and other proteins, known as the tunic. Vascular tissue is transparent and can cover several square centimeters, which is much larger than any single individual within the colony. It forms a network that ramifies and expands to the perimeter of each colony and terminates into oval-shaped protrusions known as ampullae. Botryllus individuals replace themselves through a weekly budding cycle, and vasculature is added to ensure the interconnection of each new individual, thus there is continuous angiogenesis occurring naturally. The vascular tissue itself is highly regenerative; surgical removal of the ampullae and peripheral vasculature triggers regrowth within 24-48 h, which includes forming new ampullae. When two individuals, whether in the wild or in the lab, come into close contact and their ampullae touch, they can either undergo parabiosis through anastomosing vessels, or reject vascular fusion. The vasculature is easily manipulated by direct means such as microinjections, microsurgeries, and pharmacological reagents. Its transparent nature allows for in vivo analysis by bright field and fluorescence microscopy. Here we review the techniques and approaches developed to study the different biological processes that involve the extracorporeal vasculature.